Rates of stimulant (e.g. cocaine, amphetamines) and heavy alcohol use (i.e. ?5 drinks per occasion) are significantly higher among men who have sex with men (MSM) and transgender women (TGW) than in the general population. Stimulant and heavy alcohol use are consistently associated with high-risk sexual behaviors and HIV seroconversion in MSM/TGW. Depression is also highly prevalent in this population and is known to have a strong association with substance use and HIV risk. Pre-exposure prophylaxis (PrEP) using emtricitabine-tenofovir disoproxyl fumarate (FTC-TDF) is a novel tool proven to reduce the risk of acquiring HIV. The World Health Organization and the updated US National HIV/AIDS Strategy calls for comprehensive PrEP services to dramatically reduce new HIV infection rates. However, despite high interest and uptake among MSM/TGW, over 50% disengage from PrEP within one month of starting, and it is unclear why. PrEP disengagement is defined as the discontinuation of FTC-TDF or adherence at less than 4 doses per week despite ongoing HIV risk. In several open label studies, all acute HIV seroconversions occurred exclusively during periods when PrEP was prematurely discontinued or taken suboptimally. We know from the HIV treatment literature that stimulant and alcohol use are associated with non-retention in care and non- adherence to antiretroviral therapy, but this association is not well characterized in the context of PrEP. No previous studies have utilized biomarkers of stimulant use and alcohol use to examine their relationship with PrEP disengagement. Further, no studies have evaluated the potential mechanistic effect of depression on PrEP disengagement in the context of substance use. Aims: The proposed study will address these gaps in knowledge by examining the direct associations of stimulant and heavy alcohol use on PrEP disengagement in MSM/TGW with ongoing HIV risk (Aim 1). We will also examine whether stimulant and heavy alcohol use are indirectly linked to PrEP disengagement via depressive symptoms (Aim 2). Design: The proposed study will utilize collected data and banked urine and dried blood spots from the iPrEx Open Label Extension (OLE), the largest open label study of PrEP uptake and adherence in MSM/TGW. We will test for stimulant and alcohol biomarkers to index cumulative substance use, and utilize already collected TDF drug levels and PrEP refill data to quantify disengagement. Analysis: To accomplish Aim 1, we will examine the direct associations of stimulant and heavy alcohol use on PrEP disengagement using weighted logistic regression and Cox proportional hazards. To accomplish aim 2, depressive symptomology, measured using the Center for Epidemiological Studies Depression (CES-D) scale, will be analyzed as a time-varying confounder-mediator in the causal pathway between stimulant and alcohol use and PrEP disengagement using marginal structural modeling. Results of this study will elucidate clinically relevant processes that will inform future clinical intervention studies to mitigate PrE disengagement in high-risk MSM/TGW and maximize its public health impact.